Immortalized cell lines protein production

ABSTRACT

This invention relates to the production and use of immortalized cell lines from primary chicken embryonic fibroblasts. The cells are useful as substrates for virus propagation, recombinant protein expression and recombinant virus production.

CROSS-REFERENCE TO RELATED APPLICATION

This is a division of application Ser. No. 09/151,718, filed Sep. 11,1998, (issued as U.S. Pat. No. 5,985,642 on Nov. 16, 1999), which, inturn, is a divisional of application Ser. No. 08/941,849, filed Sep. 30,1997 (issued as U.S. Pat. No. 5,879,924 on Mar. 9, 1999), which, inturn, is a continuation of application Ser. No. 08/696,200, filed Aug.13, 1996 (issued as U.S. Pat. No. 5,672,485 on Sep. 30, 1997), all ofwhich are incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to the fields of cell biology and virology. Inparticular this invention relates to the use of immortalized cells forvirus propagation.

BACKGROUND OF THE INVENTION

In 1931 Alice Miles Woodruff and Ernest Goodpasture introduced a newmethod for cultivating viruses. They reported that the virus of fowl poxcould be growvn on the chorioallantoic membrane of developing chickembryos. Lesions containing the virus appeared on the membrane aftervirus inoculation. The egg was relatively cheap and readily obtainableas compared to animals which were the substrate for early virus studies.The egg has a variety of cells and membranes susceptible to infection bydifferent viruses and can be kept in a controlled, stable environment.Chick embryos have contributed in an important way to the development ofvirology by conveniently providing a variety of cell types susceptibleto many viruses.

While the egg supports the replication of a variety of virus strains,methods for infecting the eggs and maintaining virus growth are timeconsuming and cumbersome. For example, for chorioallantoic membraneinoculation, a hole is first drilled through the eggshell and shellmembrane. The shell over the air sac is perforated causing air to enterbetween the shell membrane and the chorioallantoic membrane, creating anartificial air sac, wvhere the sample is deposited. The sample contactsthe chorionic epithelium and the virus grows as lesions on the membrane.Not unexpectedly, the use of eggs for virus replication has diminishedwith the advent of cell culture techniques.

A variety of cells can be grown in vitro. Cell cultures are easy tomaintain and can be kept in a highly controlled environment as comparedto eggs. However, there are still virus strains that appear to growbetter in embryonated egg cells than in cultured cells. In addition,many cultured cell lines carry endogenous infectious agents includingmycoplasmas, low level bacterial contaminants, endogenous viruses, andthe like. Some of the cell types that are the most efficient atsupporting virus replication have problems for viral stock production inthat the cells contain endogenous virus. The endogenous virus is eitherreplicating at a low level or can be activated when the cells areinfected with a second virus strain. For example, rodent cells are knownto carry endogenous viruses and electron microscopy of rodent cells inculture often demonstrates the existence of identifiable viral particleswithin the cells. Contaminated cell lines cannot be used as substratesfor commercial live or inactivated vaccines.

For some viruses the method of choice for viral replication is theembryonated chicken. For example, human influenza virus, rabies, CanineDistemper virus, Marek's disease virus, Reovirus and Fowl Pox virus areviruses that are preferentially grown in embryonated eggs because theegg supports high titer virus stock growth or in primary cells derivedfrom the embryonated eggs. In other cases, viruses are grown in eggsbecause there is a need for certifiable virus free cell substrates.

Primary cell cultures are cultures of cells that are freshly isolatedfrom intact tissues. These cells are often good source of virus freematerial and are well suited as host cells for virus replication.Primary cells are not always efficient at replicating virus and primaryanimal cells exhibit a limited life span in culture, eventuallyundergoing senescence. At senescence the cells cease to divide and dieout in a matter of time. The ability of cells to divide over time inculture is dependent on several parameters including the species oforigin of the cell and the age of the tissue when it was placed inculture. Cells that undergo senescence cannot be maintained in culturefor long periods of time and therefore are not useful reproducible hostsfor the growth of commercial virus stocks.

Some primary cells escape senescence and acquire the ability to becomeimmortal. Rodent cells appear to undergo spontaneous immortalizationquite easily (Curatolo et al. In Vitro 20:597-601, 1984) but normalhuman and avian cells have rarely, if ever, been shown to be capable ofspontaneous immortalization (Harvey, et al. Genes and Development5:2375-2385, 1991; Pereira-Smith, J. Cell Physiol 144:546-9, 1990; Smithet al. Science 273:63-67, 1996). There are avariety of reasons why aparticular population of cells would undergo immortalization. Cells canbe induced to undergo immortalization following exposure to agents knownto induce gene mutations. Some individuals postulate that cessation ofgrowth, related to senescence, is dominant to immortalization and eventsthat inactivate growth-restraining genes can result in immortalization(Pereira-Smith et al., Proc. Natl. Acad. Sci. (USA) 85:6042-6046, 1988).

The availability of immortalized, virus free cells can eliminate orreduce the use of primary animal tissue cultures. Primary cultures aregenerally ill-defined cell populations and are often contaminated. Thesecultures often fail to meet regulatory requirements for commercialvaccine production. Primary cultures of cells can be contaminated withCircodnavirideae (e.g., Chickenenima Virus) or Egg Drop Syndrome virus.For example, Marek's Disease vaccine (a live virus vaccine) can be grownas virus stocks in duck eggs for poultry vaccination. In 1976, flocks ofchickens receiving the vaccine showed evidence of Egg Drop Syndrome,caused by a duck adenovirus that is believed to have contaminated thevaccine stock and became adapted to growth in chickens.

In the vaccine industry, regulatory requirements for product safety,consistency and potency are driving companies to pursue cell lines asthe best alternative to the current practice of using egg-based andprimary cell vaccine substrates. Concerns for safety and consistency areshared by manufacturers of both human and animal vaccine products due toan increasingly stringent regulatory environment regarding vaccinesubstrates in both the United States and Europe. The identification ofsuitable cells for virus growth to replace embryonated eggs is alsofavored in view of US Government Principles for the Utilization and Careof Vertebrate Animals in Testing, Research, and Training and the AnimalWelfare Act (7 U.S.C. § 2131) stating, in part, that in all cases,methods such as in vitro biological systems should be considered in lieuof in vivo animal model systems. There is a need for cells that arevirus free and support exogenous virus growth to generate animal vaccineproducts.

SUMMARY OF THE INVENTION

This invention relates to the identification of spontaneouslyimmortalized chicken fibroblast cell lines and to methods for obtainingthe cell lines. In particular, this invention relates to a spontaneouslyimmortalized cell line derived from primary chicken embryonicfibroblasts having the characteristics of the spontaneously immortalizedcell line UMNSAH-DF1 that is deposited with the ATCC under the terms andconditions of the Budapest Treaty. In addition, this invention relatesto cultures of these cells and to immortalized subclones of theimmortalized cell line that support virus replication.

In one aspect of this invention the immortalized cells of this inventioncontain virus and in another the immortalized cells of this inventioncontain at least one vector capable of directing expression ofrecombinant protein in the cells. In one embodiment the cells of thisinvention express recombinant protein and in another aspect of thisinvention the vector contained in the cells of this invention encodes atleast a portion of a recombinant virus. In another embodiment the vectoris a retroviral vector.

In another aspect of this invention a method is disclosed for producingan immortalized cell line from chicken embryonic fibroblasts comprisingthe steps of: grossing primary chicken embryonic fibroblasts in culture;passaging the fibroblasts in culture until they begin cell senescence;concentrating the cells during cell senescence to maintain about 30% toabout 60% culture confluence; identifying foci of non-senescent cells;and growing the non-senescent cells for greater than 30 passages.

In yet another aspect of this invention a method is disclosed forgrowing virus in a cell comprising the steps of: growing a spontaneouslyimmortalized cell line derived from primary chicken embryonicfibroblasts in culture; infecting the cells with virus; allowing thevirus to replicate in the cells; and collecting virus that replicated inthe cells.

DETAILED DESCRIPTION OF THE PREFERRED EMDODIMENTS

At present there are essentially no non-viral, non-viral protein ornon-chemically transformed avian cell lines available. Primary celllines are cumbersome to continually generate for virus stock productionand must be separately validated as contaminant free reservoirs forvirus growth. This invention discloses the immortalization of chickenembryo fibroblastic (CEF) cells including cells derived from EastLansing Line (ELL-0) chicken embryos.

The term immortalization is used herein to refer to non-rodent cellscapable of growing in culture for greater than 30 passages that maintaina doubling time in culture of about 1 to about 2 days and have been incontinuous culture for greater than about 6 months. Avian cells aregenerally considered immortalized after about 20 to about 25 passages inculture. Immortalized cells are differentiated from transformed cells inthat unlike transformed cells, immortalized cells are density dependentand/or grown arrested (e.g., contact inhibited). Transformed cells arecapable of growth in soft agar and are usually able to form tumors wheninjected into laboratory animals. The cells of this invention are uselulas reservoirs for growing virus or for expressing recombinant protein orvirus particularly where it is important that the cells do not harborcontaminating virus or viral protein. The cells are also useful forstudying the underlying mechanisms of cellular senescence andimmortalization.

Chicken Embryo Fibroblastic (CEF) primary cells from 10 day old ELL-0eggs were obtained by taking the embryonic torso of the 10 day oldembryos, mincing the tissue and placing the cells in culture. Fertilizedeggs are available Hy-Vac (Adel, Iowa). The eggs and their layers werecertified by the supplier as negative for Avian influenza (Type A),Avian reovirus, Avian adenoviruses (Groups I-III), Avianencephalomyelitis virus, Fowl pox, Newcastle disease virus,Paramyxovirus (Type 2), Mycoplasma, Salmonella and other infectiousagents known to infect poultry stock. Isolation of primary cells andidentification of immortalized cells is provided in Example 1.

The cells were identified because at the time of the discovery of theimmortalized line, cell populations were being selected to study theeffects of cell senescence. Human and avian cells are known to be someof the most difficult cells to immortalize under tissue cultureconditions. Unlike rodent cells, there are no peer-reviewed reports ofmethods for immortalizing human or chick fibroblasts from normal donors(Smith, et al. Science 273 :63-67, 1996). In avian fibroblasts,untreated cells typically last only 20-25 passages. That is, by 30passages primary cultures of these avian cells are dead or dying. Asdisclosed in this invention, to reach 20 passages, the cells were passedand concentrated (see Example 1) between about passage 12 up to aboutpassage 20 onto smaller plates as needed. Foci of more rapidly growingcells were observed and these foci were isolated using cloning rings(Bellco Glass, Inc. Vineland, N.J.) and expanded in culture.

Senescence is defined herein as cells having population doublings ofabout 0.5 population doublings or less per day. For this inventionimmortalized cells are cells in culture for more than 30 passages,growing at a population doubling rate (as determined by total cellcounts and viable cell counts per day using trypan blue exclusion) ofabout between 0.6 to about 1.2 population doublings per day andpreferably between about 0.7 to about 1.0 population doublings per daywhile exhibiting contact inhibition, density dependence and a normalcell morphology.

The cells obtained from the originally identified foci as described inExample 1, have undergone greater than 400 (population doublings) andgreater than 160 passages. The term foci is used herein to refer toclusters of morphologically uniform cells that can be distinguished fromthe morphology of the cells around them. These foci of cells can bereadily removed and subcloned for further study. The cells of thisinvention have continued to double every 22-24 hrs. The cells werecontact inhibited, reverse transcriptase negative (see Example 2),density dependent arrested, aneuploid (as observed by chromosome spreadanalysis under oil emersion microscopy the karyotype was a mixture ofdiploid/tetraploid karyotypes with some cells displaying an apparenttranslocation of chromosome 1), and grow to high plating densities ofbetween 1.1-1.9×10⁵ cells/cm². No multinucleated giant cells wereobserved. The cells have a uniform phenotype. The cells also maintain acharacteristic pattern of rapid growth which is important for viruspropagation.

The cells were nontransformed as demonstrated by their inability to growin soft agar assays (see Example 3). In addition, the cells did notproduce tumors when injected into the wings of chickens (see Example 4).Exemplary cells of this invention were designated UMNSAH-DF1 cells andare deposited with the American Type Culture Collection (ATCC), 10801University Boulevard, Manassas, Va., 20110 as accession number CRL12203, deposited on Oct. 11, 1996 under the terms and conditions of theBudapest Treaty.

This invention also relates to the immortalized chicken embryonicfibroblast cells of this invention in culture and to subclones of theimmortalized cells of this invention. For example, the cells of thisinvention are identified as spontaneous immortalized cells. The cellsare obtained from known virus-free, known chemical contaminant-freelayers (hens producing the embryonic tissues that are the source of thisinvention) and the embryonic tissues used to produce the cells of thisinvention are also chemical contaminant-free (i.e, free from treatmentby known carcinogens or other agents known to transform rodent cells)and free from known virus. Once the immortalized cells of this inventionare in culture, it is possible to further subclone the cells to selectfor other physiological parameters that may vary in the cell populationwhile still maintaining contact inhibition and susceptibility to virusinfection.

Cells were tested for their ability to replicate HVT (Herpesvirus ofTurkeys), avian herpesvirus (serotype III), Fowl Pox virus, andreovirus. Cells can be tested for their ability to replicateCircodnavirideae, chicken HSV serotype II for a variety of other virusesand have been tested as a substrate for transfection. The cells wereuseful for propagating both avian and non-avian viruses. Example 5details methods for propagating HVT, Fowl Pox virus and reovirus. Thecells are useful as a substrate for viral production, and in particularthe cells are useful for retrovirus production since the cells and theirlayers (i.e., their mothers) did not have detectable retrovirusinfections. The cells are able to support the replication of AvianSarcoma Leukemia Virus and Rous Sarcoma Virus.

To produce virus stock, the cells of this invention can be seeded intotissue culture flasks, roller bottles, stir culture, into hollow fiberreactors or other mass culture systems. For roller bottle viruspropagation, the cells are seeded at about 2-5×10⁴ cells/cm² of surfacearea. The multiplicity of infection (ratio of infectious virus particlesto cells) to initiate virus stock growth will vary depending on virusstrain. Those skilled in the art of virology and skilled in the growthof particular viruses and strains of viruses will be able to maximizevirus stock yield through the standard manipulation of the multiplicityof infection, temperature, media variations, and the like, without undueexperimentation.

Methods for harvesting the virus after infection to obtain infectiousvirus stock also varies with virus strain. Enveloped viruses egress intothe culture media more slowly than non-enveloped virus. Stocks of viruscan be obtained from the culture media alone or from cell lysates pooledwith the conditioned media. For lytic viruses (those efficient at lysinga cell during virus egress), harvesting the conditioned culture media(e.g., spent media containing virus) after a gentle centrifugation stepto remove cell debris is sufficient. Again, methods for harvesting andsaving virus from a wide range of virus strains are well known in the

There are a variety of methods, also all known in the art, forquantitating virus growth from a culture of cells. For example, thetiter of a virus stock for members of the Herpesvirus family and for avariety of viruses producing foci of cytopathology on a cell monolayersurface are readily quantitated by plaque assay (as plaque formingunits/ml of culture fluid or as plaque forming units/dose for vaccineinoculum virus quantitation) or as tissue culture infectious dose-50(TCID₅₀). Rapidly lytic viruses are better quantitated by TCID₅₀ as thedose or dilution of virus stock capable of infecting 50% of the culturesin a defined time period. Methods for growing and quantitating virus areknown in the art and sources for teaching virus quantification methodsare found in Fields, et al. (eds) Fundamental Virology 1991, RavenPress, New York or in Mandell, et al. (eds.) Principles and Practiceoflnfectioits Diseases, 1985, John Wiley & Sons, New York.

In addition to supporting virus growth, the cells of this invention canbe used as packaging lines to produce recombinant virus, includingretrovirus. The cells can also be used to produce recombinant proteins,including viral proteins, andthe like. Methods for incorporating nucleicacid encoding recombinant protein into a nucleic acid vector under thecontrol of regulatory elements capable of directing expression of aprotein in a eukaryotic cell, such as the immortalized cells of thisinvention, are well known in the art. Expression vectors are replicablenucleic acid fragments that can direct expression of a recombinantprotein. Many expression vectors, including retroviral vectors, areavailable in the art through journal publications and commercialsuppliers. Replicable expression vector components generally include,but are not limited to, one or more of the folloing: an origin ofreplication, one or more marker genes, enhancer elements, promoterelements, optional signal sequences and transcription terminationsequences. The selection or marker genes encode protein that serves toidentify a population of transformed or transfected cells. Typicalselection genes encode proteins that confer resistance to antibiotics orother toxins, complement auxotrophic deficiencies or supply criticalnutrients not available from complex media.

Expression vectors having nucleic acid encoding recombinant protein aretransfected into the cells and are used to direct expression of therecombinant protein in the immortalized cells of this invention. Thevector preferably can encode any recombinant protein capable ofexpression in chicken embryonic fibroblast cells, including, but notlimited to, virus protein, including reverse transcriptase and/or viralstructural protein. Examples of vectors to produce recombinant proteinin a cell include retroviral vectors to produce tumor suppressiveprotein, or viral structural protein such as those disclosed by Givol,et al. Oncogene 11(12):2609-2618, 1995, Givol, et al. Cell Growth &Differentiation 5(4):419-429, 1994, Federspiel, et al. Virology203(2):211-220, 1994 and Boyer, et al. Oncogene 20:457-66, 1993.

The cells of this invention can serve as substrate to expressrecombinant virus, including, but not limited to recombinant retrovirus.The cells of this invention are suitable to serve as packaging celllines for genetically engineered virus useful for gene therapy, or thelike. Constructs and methods for using a particular cell line as apackaging cell line are known in the art. For example, Boerkoel, et al.(Virology 195(2):669-79, 1993) discloses methods for packaging virususing primary chicken embryonic fibroblasts as the packaging cell line.These same methods can be used to package virus in the immortalizedcells of this invention.

Since most avian cell lines and all transformed avian cells as well asvirtually all mouse transformed cell lines either contain viralcontaminants such as endogenous virus or produce viral protein, they arenot suited for the production of human or animal vaccines. The cellscannot be used to produce recombinant protein because the endogenouscontaminants can contaminate purified recombinant protein preparations.Advantageously, the cells of this invention provide a suitablealternative to these problems.

The cells of this invention can also serve as a substrate for supportingvirus growth from other cells. These other cells include primary cells,or cultured cells that show improved growth or longevity in culture inthe presence of other cells or in the presence of extracellular matrixproteins such as collagens, laminins, and the like. In one embodiment,cells are mixed with virus and then mixed faith the cells of thisinvention preferably in a ratio of cells: to cells of this invention ofabout between 1:5 cells to about 1:20 cells and more preferably in aratio of about 1:10 (1 cell to about 10 cells of this invention). Themixed cells are then placed into culture. In a second embodiment thecells are mixed with virus and plated onto the surface of theimmortalized cells of this invention are already attached to a tissueculture surface. The cells of this invention serve as a support for theother cells and, without intending to limit the scope of this invention,the cells of this invention can supply growth factors and the like aswell as extracellular matrix components, and the like to support theother cells while they are producing virus. Example 6 provides anexample of the use of the cells of this invention as a cell substrate.

Particular embodiments of this invention will be discussed in detail andreference has been made to possible variations within the scope of thisinvention. There are a variety of alternative techniques and proceduresavailable to those of skill in the art which would similarly permit oneto successfully perform the intended invention.

EXAMPLE 1 Establishment of Spontaneous Chicken Fibroblast Cell Line

Two dozen ELL-0 eggs wvere ordered from East Lansing USDA poultrystocks. The eggs were incubated in a sterilized isolated incubator for10 days and were processed for primary cultures. Embryonic tissue vasdissociated using a trypsin/EDTA solution and plated in DMEM media(Gibco) containing 10% fetal calf serum (Gibco). 1%antibiotic/antimrncotic (Gibco) containing and 2 mM L-glutamine (Gibco).The dissociated cell suspension was collected in a 50 ml centrifuge tubecontaining 10% ml fetal bovine serum to inactivate the trypsin andcentrifuged at 700×g for 10 minutes.

The cells were resuspended in 10 ml Dulbecco's modified Eagles's mediumenriched with 36 μg/ml insulin (Sigma), 1.6 μg/ml transferrin (Sigma,St. Louis, Mo.), 2 mM L-glutamine, 10% fetal calf serum, 1%antibiotic/antimycotic solution and pipetted into a 25 cm² coming tissueculture flask and incubated at 40.5° C. in 5% CO₂, 95% air. After 24hours of incubation, the media was changed. The priary culture containednumerous explants with centers of epithelial-like cells and radiatingfibroblasts.

Cultures were allowed to grow to confluency (5 days) and were removedfrom the plates using a trypsin/EDTA solution (0.05% trypsin and 0.02%ethylene diamine tetra acetic acid (EDTA) in PBS) and replated forsecond passage. At second passage some of the cells were frozen in aconditioned media containing 50% DMEM media, 12% DMSO and 38% fetal calfserum. These cells were frozen in the vapor phase liquid nitrogen for 24hours then transferred to the aqueous liquid nitrogen for long termstorage.

Cells at second passage (P2) were replated at a seeding density of2.7×10⁴ cells/cm². The cells were sub-cultured for several months. Thecultured fibroblasts grew rapidly for 8 to 9 passages, then began toslow down with significant cell death. During crises, the cells wverepassed using an ATV solution (8 gm/l NaCl, 0.4 gm KCl, 1 gm dextrose,0.58 gm NaHCO₃, 0.5 gm trypsin (Difco 1:250), 0.2 gm versene (disodiumnsalt) in 1000 mL). Cells were grown in Dulbecco's modified Eagles'smedium enriched with 36 μg/ml insulin (Sigma), 1.6 μg/ml transferrin(Sigma), 2 mM L-glutamine, 10% fetal calf serum and 1%antibiotic/antimycotic solution. It was noted that the majority of thecells at passage 11 (P11) were dead or dying; however, a smallsubpopulation of cells appeared to be healthy fibroblasts. The P11 cellsremained on the dish for four weeks with refeeding every three days withfresh media. Some cells were frozen and the remaining cells wereconcentrated into a smaller area and were allowed to grow another twoweeks before they were confluent enough for a second subculturing. ByP15, the cells were appearing to be more homogeneous in cellularmorphology and were growing at a rate of 0.32 population doublings perday. By P20, the population doublings increased to about 0.7 to about0.8 population doublings per day. At this time the cells appeared tohave a very uniform morphology. The cells were denoted UMNSAH/DF #1 andhave been in continuous culture for over nineteen months. The cells arecurrently at passage 160. Cells were frozen (as above) and thawed fromP5. The subcloned cells were expanded and the reproducibility of themethod was confirmed through the identification of other clones. Severalmore subclones were obtained by P11.

EXAMPLE 2 Testing Cells for Virus Contaminants

The cells of this invention are tested for viral contaminants using PCRto identify contaminating nucleic acid fragments. There are a widevariety of commercially available test kits for a variety of virusesthat can be used to determine whether the cells of this inventioncontain contaminating virus. Similarly, there are commercially availabletests to detect viral antigen (e.g., commercially available ELISA assaysand the like), where the antigen is derived from a variety of differentviruses. These tests can be used on the cells of this invention usingroutine experimental techniques to demonstrate that the cultures arefree of contaminating virus.

In one series of tests, the cells were tested for reverse transcriptaseactivity. 1×10⁶ cells from rapidly growing cultures were isolated in 4ml. of media. The media was taken through several freeze thaws at −80°C. to lyse the cells. The media with lysed cells were layered over a 10%glycerol gradient. The gradient was spun for 60 minutes at 40,000 rpmusing an SW40 rotor (Beckmran Instruments, Palo Alto, Calif.). Virusparticles, if present were pelleted. The media was discarded and thepellet was resuspended in 20 μl of Nonidet P-40 (Sigma Chemical Co., St.Louis, Mo.).

An eppendorf tube was heated at 41° C. 5 μl of sample was added to 45 μlof reverse transcriptase cocktail containing 45 mM Tris, pH 7.8, 2 mM2-β mercaptothanol, 2 mM manganous acetate, 0.1% Triton X-100, 10 μMeach dATP, dCTP, dGTP (Boehringer Mannheim Biochemical, Indianapolis,Ind.), 2.4 μg polyA (Sigma), 60 ng primer dT 12-19 (Pharmacia), 0.4μCi/reaction ³H thymidine triphosphate (15,000 to 28,000 cpm/pmoleactivity, Amersham).

The reaction was incubated for one hour at 41° C. A negative controlincluded 5 μl of ddH₂O and 45 μl of the cocktail. Two known positivecontrols were included with the assay. The assay was stopped by adding 1ml of 10% trichloroacetic acid (TCA, Columbus Chemical Industries, Inc.,Columbus, Wis.). The mixture wvas filtered through a Whatman GF/C glass0.45 micron pre filter. Several washes were performed using 5% TCA. Thefilter was transferred to a Beckman Instruments Scintillation Counterusing scintillation vials containing 5 mls of scintillation countingfluid. Samples were counted on a 050 to 600 window setting. An increaseof threefold counts over the cocktail background (neg. control) wasconsidered positive.

The primary cultures tested negative for reverse transcriptase as didthe immnortalized cells obtained in this invention. For furtherinformation on reverse transcriptase assays see (Crittenden, et al.Virology 57:128-138, 1974).

EXAMPLE 3 Soft Agarose Colony Formations Assay to Assess TumorigenicPotential of Cells

To test for tumorigenic potential, the cells were tested for growth insoft agar. A soft agarose base was made by mixing 12 ml of a 2% agarosesolution (that had been autoclaved and cooled to 56° C.) in 21.6 mls ofenriched McCoy's 5A medium [Gibco, 120 mls fetal calf serum (heatinactivated, 5 mls Na pyruvate (2.2% stock), 1 ml L-serine (21 mg/mlstock), 5 mls L-glutamine (200 mM stock), 12.5 mls Hepes (1M stock)],5.9 mls Asparagine (4.4 mg/ml filtered sterilized stock). Seven mls ofwarm medialagarose was poured onto a 100 mm² tissue culture dish andallowed to solidify at room temperature in a tissue culture hood for 1hr.

Cells were removed from actively growing cultures (abbut 40% to about70% confluent) by trypsinization to achieve a single cell suspension infresh DMEM media containing 10% fetal calf serum (with L-glutamine andantibiotics-antimycotic). Approximately 1×10⁶ cells was added to 4.25 mlof DMEM media containing 10% fetal calf serum, 0.75 ml of 1% agarose,and 50 μl 2β-mercaptoethanol. Care was needed to be certain that thewarm media/agarose was at 42° C. before adding the cells. Quickly, 5 mlof the above cell suspension was overlaid on the agarose plates.

Cells were growvn at 37° C. in a 5% CO₂ 95% air incubator and observedfor 35 days. Duplicate plates were stained with 3 p-nitrophenyl-5-phenyltetrazolium chlorite (INT stain) and examined at days 0,5,10,15,20,30and 35 for colony formation and growth. All stained colonies greaterthan 60 μm were considered positive.

All cells tested negative. Further information related to the soft-agarassay is available from Hamburger et al., Prog. Clin. Biol. Res.,Cloning of Human Tumor Stem Cells, 48, 43-52 (1980); S. Salmon, Prog.Clin. Biol. Res., Cloning of Human Tumor Stem Cells, 135-151 (1980); andB. Kressner et al., Prog. Clin. Biol. Res., Cloning of Human Tumor StemCells, 179-193 (1980).

EXAMPLE 4 Tumorgenicity of Immortalized Cells

Under the guidelines outlined in the University of Minnesota AnimalUsage Protocol (protocol #950300-1, March 1995-December 1996) cells wereinjected into test animals to determine whether or not the cells weretumorigenic.

Actively grooving cells were removed from cell culture plates and werenjected into six SPAFAS line adult chickens (Hy-Vac, Adel, Iowa).Subcutaneous injections of 4×10⁶ cells were introduced into the wingwebs of the chickens. The sites of injection were examined weekly for3.5 months. No tumors were observed at the injection site for any of thetransfected cells produced to date with all animals remaining healthy.The experiment demonstrated that the immortalized cells werenontumorigenic.

EXAMPLE 5 Ability of Cells to Support Virus Growth

The cells were seeded into roller bottles at 5.0×10³ cells/cm². Thecells were allowed to attach for 24 hours and a control was harvestedfor cell counts. Cells were grown for virus infection in DMEM (4.5 g/Lglucose), 4% Fetal Bovine Serum, 2 mM L-Glutamine, 50 mg/L Gentamicin.Cells were infected at a multiplicity of infection of 0.0006 HVT virusparticles per cell. The roller bottles were watched daily forprogression of CPE. The bottles were harvested at 46 hs. post infectionwhen there was approximately 50% CPE. HVT infected cells were frozen ingrowth medium with 10% DMSO at a concentration of 2.0×10⁷ cells/ml.Titers of HVT were quantitated by plaque assay. Virus was seriallydiluted in growth media and placed onto confluent monolayers ofpermissive cells. Cultures were incubated for a designated time and thecells were fixed and stained. Plaques on the monolayers wvere countedand virus titer was expressed as plaque forming units per dose.

These cells were also tested for their ability to support reovirusproduction. 2.5×10³ cells were infected with WSS-Reo 1733 strain ofReovirus having a titer of 8.2 TCID₅₀/ml. Cells were infected at amultiplicity of infection of 0.005, 0.001 or 0.0005 infectious virusparticles/cell. Infected cells wvere grown in roller bottles and testedat 48, 64 and 72 hours after infection to demonstrate productive viralgrowth.

EXAMPLE 6 Use of Transfected Skin Cells as a Cell Substrate

The cells of this invention are useful as a substrate for supportingvirus replication of primary cells. In these experiments theimmortalized cells are mixed with primary cells. In one study theprimary cells are infected and mixed with the immortalized cells andplaced in culture and in another study the primary cells are infectedand placed onto the immortalized cells where the immortalized cells arealready positioned as a lawvn in the tissue culture flask. In oneexample the virus is Egg Drop Syndrome virus and the primary cells areprimary chicken embryonic liver cells. In a second example the primarycells are endothelial cells, preferably kidney endothelial cells and thevirus is infectious bronchitis virus. The preferred ratio of primarycells to immortalized cells is about 1:5 to about 1:20 and morepreferably about 1:10. Virus titers from primary cells growing in themixed cell population are higher than virus titers from primary cells inculture alone. The immortalized cells allow the primary cells to be usedfor virus propagation under commercial conditions.

All cited publications are incorporated by reference in their entiretyinto this text. Although the invention has been described in the contextof particular embodiments, it is intended that the scope of coverage ofthe patent be limited only by reference to the following claims.

What is claimed is:
 1. A method for producing protein in an immortalized cell comprising the steps of: obtaining immortalized chicken cells by: growing primary chicken embryonic fibroblasts in culture, passaging the fibroblasts in culture until they begin cell senescence, concentrating the cells during cell senescence to maintain about 30% to about 60% culture confluence, identifying foci of non-senescent cells in the culture, isolating the non-senescent cells, and growing the non-senescent cells for greater than 30 passages; introducing nucleic acid encoding at least one protein into the cells; and isolating protein from the cells.
 2. The method of claim 1 wherein the nucleic acid encoding at least one protein is present in a vector.
 3. The method of claim 1 wherein the obtaining step comprises obtaining cells identified as ATCC deposit number CRL-12203.
 4. The method of claim 2 wherein the gene vector is a retroviral vector.
 5. Immortalized chicken cells producing protein according to claim
 1. 6. A method for growing cells comprising the steps of: placing a first cell into cell culture with immortalized chicken cells, the immortalized chicken cells obtained by: growing primary chicken embryonic fibroblasts in culture, passaging the fibroblasts in culture until they begin cell senescence, concentrating the cells during cell senescence to maintain about 30% to about 60% culture confluence, identifying foci of non-senescent cells in the culture, isolating the non-senescent cells, and growing the non-senescent cells for greater than 30 passages maintaining the first cell in culture.
 7. The method of claim 6 wherein the immortalized cells are cells identified as ATCC deposit number CRL-12203.
 8. A spontaneously immortalized cell line, derived from primary chicken embryonic fibroblasts, wherein the cell line is capable of growth in culture at a population doubling rate of about between 0.6 to 1.2 population doublings per day. 